Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal) function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases

High fidelity copy number analysis of formalin-fixed and paraffin-embedded tissues using affymetrix cytoscan HD chip

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples. © 2014 Yu et al

The degree of segmental aneuploidy measured by total copy number abnormalities predicts survival and recurrence in superficial gastroesophageal adenocarcinoma

Background: Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC. Methods: We quantified copy number changes in 41 superficial EAC using Affymetrix SNP 6.0 arrays. We identified recurrent chromosomal gains and losses and calculated the total copy number abnormality (CNA) count for each tumor as a measure of aneuploidy. We correlated CNA count with overall survival and time to first recurrence in univariate and multivariate analyses. Results: Recurrent segmental gains and losses involved multiple genes, including: HER2, EGFR, MET, CDK6, KRAS (recurrent gains); and FHIT, WWOX, CDKN2A/B, SMAD4, RUNX1 (recurrent losses). There was a 40-fold variation in CNA count across all cases. Tumors with the lowest and highest quartile CNA count had significantly better overall survival (p = 0.032) and time to first recurrence (p = 0.010) compared to those with intermediate CNA counts. These associations persisted when controlling for other prognostic variables. Significance: SNP arrays facilitate the assessment of recurrent chromosomal gain and loss and allow high resolution, quantitative assessment of segmental aneuploidy (total CNA count). The non-monotonic association of segmental aneuploidy with survival has been described in other tumors. The degree of aneuploidy is a promising prognostic biomarker in a potentially curable form of EAC. © 2014 Davison et al

High MIG (CXCL9) plasma levels favours response to peginterferon and ribavirin in HCV-infected patients regardless of DPP4 activity

Sustained virological response (SVR) following peginterferon (pegIFN) and ribavirin (RBV) treatment in hepatitis C virus (HCV) infected patients has been linked with the IL28B genotype and lower peripheral levels of the CXCR3-binding chemokine IP-10 (CXCL10). To further improve the understanding of these biomarkers we investigated plasma levels of the other CXCR3-binding chemokines and activity of the dipeptidyl peptidase IV (DPP4, CD26) protease, which cleaves IP-10, in relation to treatment response

cn.FARMS: a latent variable model to detect copy number variations in microarray data with a low false discovery rate

Cost-effective oligonucleotide genotyping arrays like the Affymetrix SNP 6.0 are still the predominant technique to measure DNA copy number variations (CNVs). However, CNV detection methods for microarrays overestimate both the number and the size of CNV regions and, consequently, suffer from a high false discovery rate (FDR). A high FDR means that many CNVs are wrongly detected and therefore not associated with a disease in a clinical study, though correction for multiple testing takes them into account and thereby decreases the study's discovery power. For controlling the FDR, we propose a probabilistic latent variable model, ‘cn.FARMS’, which is optimized by a Bayesian maximum a posteriori approach. cn.FARMS controls the FDR through the information gain of the posterior over the prior. The prior represents the null hypothesis of copy number 2 for all samples from which the posterior can only deviate by strong and consistent signals in the data. On HapMap data, cn.FARMS clearly outperformed the two most prevalent methods with respect to sensitivity and FDR. The software cn.FARMS is publicly available as a R package at http://www.bioinf.jku.at/software/cnfarms/cnfarms.html

Plasma levels of growth-related oncogene (CXCL1-3) associated with fibrosis and platelet counts in HCV-infected patients

Fibrosis progression in hepatitis C virus (HCV)-infected patients varies greatly between individuals. Chemokines recruit immune cells to the infected liver and may thus play a role in the fibrosis process

Assessing HER2 gene amplification as a potential target for therapy in invasive urothelial bladder cancer with a standardized methodology: results in 1005 patients

Background: This study assessed the human epidermal growth factor receptor-2 (HER2) protein expression and its relationship with gene amplification in invasive bladder carcinoma, using the same criteria than for breast cancer

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

BACKGROUND:A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.RESULTS:Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.CONCLUSION:MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]